A cello-oligosaccharide is an oligosaccharide formed by polymerization of two to six glucose molecules and is a linear oligosaccharide in which two to six glucopyranose units are linked by β-1,4-glycosidic bonds. It is anticipated that cello-oligosaccharides will serve as materials that can be used in the fields of functional foods, energy, feed, chemical engineering products or the like.
In a known method for producing cello-oligosaccharides, decomposition of cellulose as a starting material by cellulases is utilized. As shown in FIG. 1, the mechanism of decomposition of cellulose by cellulases includes (1) reaction where cello-oligosaccharides including cellobiose are produced from cellulose by the effect of cellulose degrading enzymes, (2) reaction where glucose is produced from cellulose and (3) reaction where glucose is produced from cello-oligosaccharides by the effect of a β-glucosidase (NPL 1).
As shown in FIG. 1, there are three types of enzymatic activity for cellulases: endoglucanases which randomly decompose cellulose (sometimes abbreviated to EG below); cellobiohydrolases which release cellobiose from the ends of cellulose chains (sometimes abbreviated to CBH below); and β-glucosidases which decompose cellobiose into two glucose molecules (sometimes abbreviated to BGL below).
In order to increase the productivity of cello-oligosaccharides, it is effective to inhibit the decomposition of cello-oligosaccharides by inhibiting a β-glucosidase contained in cellulases and inhibiting the decomposition of the cello-oligosaccharides into glucose. With respect to methods for inhibiting a β-glucosidase contained in cellulases, various attempts including enzyme fractionation using fractionation by chromatography shown in FIG. 2 have been made.
PTL 1 discloses a method for selectively producing cellobiose from cellulose using cellulases obtained by adsorbing cellulases onto a weak-acid cation exchange resin which has been equilibrated at pH 3.5 to 5.0 to remove a β-glucosidase from the cellulases.
PTL 2 discloses a method for producing cello-oligosaccharides by adsorbing the enzyme components contained in cellulases, except for β-glucosidases, onto cellulose or a cellulose-containing substance in advance and then conducting enzymolysis.
PTL 3 describes a method for separating and removing a β-glucosidase from cellulases characterized by filling a tube or tower reactor with a cellulosic material, letting a cellulase solution that contains a cellobiohydrolase and contains, as impurities, a β-glucosidase continuously pass through the reactor in one direction and thus selectively separating and removing the fraction containing the β-glucosidase.
These conventional methods can reduce the amount of β-glucosidase that contaminates during the production step of cellobiose, and cellobiose can be obtained selectively. However, these methods require a step of adsorbing cellulases onto a solid fraction and a step of separating the solid fraction and thus have problems because the process is complex and costs are high.
In addition, a method for inhibiting the β-glucosidase activity has been developed, and PTL 4 describes a method for producing cellobiose in which δ-gluconolactone or gluconic acid, which is a β-glucosidase inhibitor, is made exist during the decomposition of cellulose by cellulases.
In the method described in PTL 5, glucose oxidase is made exist during the decomposition of cellulose by cellulases and δ-gluconolactone is produced from the produced glucose, and as a result it is attempted to inhibit the β-glucosidase activity.
However, the practical application of these methods has been difficult because of the complexity of the separation of cellobiose as the product from δ-gluconolactone, high price of δ-gluconolactone and the like.